Project Summary Staphylococci and C. albicans are the most frequent combination of organisms isolated from polymicrobial infections. The ability of C. albicans and S. aureus to cause disease largely depends on their ability to form biofilms and alter their transcriptome in response to different stresses to ensure survival in the host. Changes in the transcriptional network also ensure that these organisms can grow in tissues and survive stresses inflicted upon them by cells of the innate immune system. Significantly, enhanced in vitro tolerance to antimicrobials in C. albicans and staphylococci mixed biofilms has been reported. Consequently, in addition to formation of enhanced and persistent biofilms, co-infection with S. aureus and C. albicans may influence gene expression, in turn impacting virulence and drug resistance. Therefore, it has become imperative to elucidate the unique interactions between these pathogens within polymicrobial biofilms in vivo with the goal of providing overall insights into the dynamics of how mixed- microbial populations are established and regulated in the human body. To that end, we developed a practical and clinically-relevant subcutaneous catheter mouse model to characterize the molecular interplay between S. aureus and C. albicans as they co- infect a host by performing comparative transcriptomics analysis of single and mixed- species biofilms. Further, we designed feasible non-invasive bioluminescence imaging and intravital microscopy systems as tools, which we will use to elucidate the mechanisms of biofilm formation and development of drug resistance under in vivo conditions. Our central hypothesis is that the regulatory and transcriptional pathways uniquely expressed during in vivo-grown polymicrobial biofilms of C. albicans and S. aureus identify microbial phenotypes of enhanced pathogenic potential central to the persistence and antimicrobial resistance of biofilm-associated polymicrobial infections.